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Molecular Basis for Enantioselectivity of Lipase from Chromobacterium viscosum toward the Diesters of 2,3-Dihydro-3-(4′-hydroxyphenyl)-1,1,3-trimethyl-1H-inden-5-ol David G. Gascoyne,† Herman L. Finkbeiner,† Kwok P. Chan,† Janet L. Gordon,† Kevin R. Stewart,† and Romas J. Kazlauskas*,‡ Molecular OptoElectronics Corporation, 877 25th Street, Watervliet, New York 12189, and McGill University, Department of Chemistry, 801 Sherbrooke Street West, Montre´ al, QC H3A 2K6 Canada
[email protected] Received October 12, 2000
2,3-Dihydro-3-(4′-hydroxyphenyl)-1,1,3-trimethyl-1H-inden-5-ol, 1, is a chiral bisphenol useful for preparation of polymers. Previous screening of commercial hydrolases identified lipase from Chromobacterium viscosum (CVL) as a highly regio- and enantioselective catalyst for hydrolysis of diesters of 1. The regioselectivity was g30:1 favoring the ester at the 5-position, while the enantioselectivity varied with acyl chain length, showing the highest enantioselectivity (E ) 48 ( 20 S) for the dibutanoate ester. In this paper, we use a combination of nonsymmetrical diesters and computer modeling to identify that the remote ester group controls the enantioselectivity. First, we prepared nonsymmetrical diesters of (()-1 using another regioselective, but nonenantioselective, reaction. Lipase from Candida rugosa (CRL) showed the opposite regioselectivity (>30:1), allowing removal of the ester at the 4′-position (the remote ester in the CVL-catalyzed reaction). Regioselective hydrolysis of (()-1-dibutanoate (150 g) gave (()-1-5-dibutanoate (89 g, 71% yield). Acylation gave nonsymmetrical diesters that varied at the 4′-position. With no ester at the 4′-position, CVL showed no enantioselectivity, while hindered esters (3,3-dimethylbutanoate) reacted 20 times more slowly, but retained enantioselectivity (E ) 22). These results indicate that the remote ester group can control the enantioselectivity. Computer modeling confirmed these results and provided molecular details. A model of a phosphonate transition state analogue fit easily in the active site of the open conformation of CVL. A large hydrophobic pocket tilts to one side above the catalytic machinery. The tilt permits the remote ester at the 4′-position of only the (S)-enantiomer to bind in this pocket. The butanoate ester fits and fills this pocket and shows high enantioselectivity. Both smaller and larger ester groups show low enantioselectivity because small ester groups cannot fill this pocket, while longer ester groups extend beyond the pocket. An improved large-scale resolution of 1-dibutanoate with CVL gave (R)-(+)-1-dibutanoate (269 g, 47% yield, 92% ee) and (S)-(-)-1-4′monobutanoate (245 g, 52% yield, 89% ee). Methanolysis yielded (R)-(+)-1 (169 g, 40% overall yield, >97% ee) and (S)-(-)-1 (122 g, 36% overall yield, >96% ee). Introduction Organic materials with nonlinear optical properties are the recent focus of both industrial and academic research due to the potential applications in telecommunications, optical information processing, storage and display.1 Many nonlinear optical effects require a noncentrosymmetric, that is, chiral, environment for the chromophore. One way to create a chiral environment is to align the chromophore in poled polymer films or LangmuirBlodgett films. A second way is to add chiral substituents to the chromophore. A third way is to create a chiral supramolecular array, such as a chiral liquid crystal or a chiral polymer. Devices with the strongest nonlinear optical effect often use several methods. For example, a Langmuir-Blodgett film of helicene showed a small nonlinear optical effect, but this effect increased ∼30* To whom correspondence should be addressed at McGill University. Phone: +1 514 398 7229, fax: +1 514 398 3797. † Molecular OptoElectronics Corporation. ‡ McGill University. (1) Example: Nalwa, H. S.; Miyata, S., Eds. Nonlinear Optics of Organic Molecules and Polymers; CRC: Boca Raton, FL, 1997.
fold when researchers used pure enantiomers of helicene in the film.2 The pure enantiomers created a chiral supramolecular array similar to a liquid crystal. In another example, researchers used all three approaches. They added a chiral substituent to a chromophore, oriented it into a liquid crystal, poled it, and finally crosslinked this orientation.3 To use chiral polymers as supramolecular arrays in nonlinear optics requires polymers with good transparency and mechanical properties because they also form the mechanical shape of the device. Chiral polymers derived from polyacrylates or polystyrene with added chiral side chains4 have good transparency and mechanical properties, but the stereocenter lies far from the main chain. Chiral polymers with a stereocenter in the main chain would probably better control the polymer chain (2) Verbiest, T.; van Elshocht, S.; Kauranen, M.; Hellemans, L.; Snauwaert, J.; Nuckolls, C.; Katz, T. J.; Persoons, A. Science 1998, 282, 913-915. (3) Trollsås, M., Orrenius, C., Sahle´n, F., Gedde, U. W., Norin, T., Hult, A., Hermann, D., Rudquist, P., Komitov, L., Lagerwall, S. T., Lindstro¨m, J. J. Am. Chem. Soc. 1996, 118, 8542-8548.
10.1021/jo005681v CCC: $20.00 © 2001 American Chemical Society Published on Web 04/12/2001
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Scheme 1. Carbenium Ion Mediated Dimerization of 4-(2-Propenyl)phenol Yields Racemic 2,3-Dihydro-3(4′-hydroxyphenyl)-1,1,3-trimethyl-1H-inden-5-ol, (()-1, a Bisphenol Monomer Useful for the Synthesis of Polymers
orientation. The ideal monomer would be rigid, contain a stereocenter within the main polymer chain, and form transparent polymers with excellent mechanical properties. Unfortunately, no such examples exist. One group recently reported binaphthol polycarbonates, but their molecular weights were very low.5 A monomer that fits these criteria is Indane bisphenol dimer or 2,3-dihydro-3-(4′-hydroxyphenyl)-1,1,3-trimethyl1H-inden-5-ol, 1. Acid-catalyzed dimerization of 4-propenylphenol,6 Scheme 1, yields racemic 1 in high yield. It also forms as an impurity in the commercial preparation of bisphenol A (2,2-bis(4-hydroxyphenyl)propane).7 Racemic 1 forms transparent polymers with good mechanical properties.8 We previously reported a regio- and enantioselective hydrolysis of diesters of 19 using lipase from Chromobacterium viscosum.10 Although chemists often resolve enantiomers using hydrolase-catalyzed reactions,12 this reaction was unusual for two reasons. First, the hydrolysis was highly regio- and enantioselective despite the large distance between the stereocenter and ester car(4) For example, Chen, X. H.; Herr, R. P.; Schmitt, K.; Buchecker, R. Liq. Cryst. 1996, 20, 125-138; Firestone, M. A.; Park, J.; Minami, N.; Ratner, M. A.; Marks, T. J.; Lin, W.; Wong, G. K. Macromolecules 1995, 28, 2247-2259. (5) Takata, T.; Furusho, Y., Murakawa, K.; Endo, T., Matsuoka, H.; Hirasa, T.; Matsuo, J.; Sisido, M. J. Am. Chem. Soc. 1998, 120, 45304531. (6) Chan, K. P.; Cristo, P. L.; McGrath, P. M. US Patent 5,994,596, 1999. (7) Paryzkova, J.; Snobl, D.; Matousek, P. Chem. Prum. 1979, 29, 30-34. (8) For example, polycarbonates: Gordon, J. L.; Chan, K. P.; Gascoyne, D. G. Polym. Prepr. 1998, 39, 486-487; Gordon, J. L.; Gascoyne, D. G. US Patent 5,703,197, 1997; polyethers: Saegusa, Y.; Iwasaki, T., Nakamura, S. J. Polym. Sci., Part A: Polym. Chem. 1994, 32, 249-256; polysilixoanes: Saegusa, Y.; Kato, T.; Oshiumi, H.; Nakamura, S. J. Polym. Sci., Part A: Polym. Chem. 1992, 30, 14011406; Padmanaban, M.; Kakimoto, M.; Imai, Y. J. Polym. Sci., Part A: Polym. Chem. 1990, 28, 2997-3005; polyesters: Wilson, J. C. J. Polym. Sci., Polym. Chem. Ed. 1975, 13, 749-754; Imai, Y.; Tassavori, S. J. Polym. Sci., Polym. Chem. Ed. 1984, 22, 1319-1325; Kakimoto, M.; Harada, S.; Oishi, Y.; Imai. Y. J. Polym. Sci., Part A: Polym. Chem. 1987, 25, 2747-2753; Negi, Y. S.; Imai, Y.; Kakimoto, M. J. Polym. Mater. 1988, 5, 67-71; Yoneyama, M.; Kakimoto, M.; Imai, Y. Macromolecules 1989, 22, 2593-2596; polyphosphates: Imai, Y.; Kamata, H.; Kakimoto, M. J. Polym. Sci., Polym. Chem. Ed. 1984, 22, 1259-1265. (9) Zhang, M., Kazlauskas, R. J. Org. Chem. 1999, 64, 7498-7503; Kazlauskas, R. J.; Zhang, X. U.S. Patent 5,959,159; 1999. (10) Lipases from Chromobacterium viscosum and from Pseudomonas glumae have identical amino acid sequences and very similar biochemical properties. Taipa, M. A.; Liebeton, K.; Costa, J. V.; Cabral, J. M. S.; Jaeger, K.-E. Biochim. Biophys. Acta 1995, 1256, 396-402; Lang, D. Hofmann, B. Haalck, L. Hecht, H. J. Spener, F. Schmid, R. D. Schomburg, D. J. Mol. Biol. 1996, 259, 704-717. (11) (a) Faber, K. Biotransformations in Organic Chemistry, 4th ed.; Springer: Berlin, 2000. (b) Bornscheuer, U. T.; Kazlauskas, R. J. Hydrolases in Organic Syntheses: Regio- and Stereoselective Biotransformations Wiley-VCH: Weinheim, 1999. (c) Wong, C.-H.; Whitesides, G. M. Enzymes in Synthetic Organic Chemistry, Pergamon: Oxford, 1994.
Table 1. Influence of the Acyl Group on the Enantioselectivity of CVL toward 1-Diesters
ester
rate (U/mg)
conversion (%)a
ee of monoester (%)b
Ec
diacetate dipropanoate dibutanoate dipentanoate dihexanoate diheptanoate dioctanoate dinonanoate didecanoate
Straight Chain 15 63d 11.5 37 8 36 11 39 0.4 51 10 3.4 2.8 8.8 6.4 47 6.8 20
42.3 78.5 93.2 92.4 78.4 86.4 63.8 51.1 60.1
5 14 ( 3 48 ( 20e 35 ( 18e 21 12 5 5 5
2-methylbutan-oate 3-methylbutan-oate 3,3-dimethyl-butanoate
Branched Chain 2 18 97% ee
1 2 3 4 total
(S)-(-)-1 79.8 40.4 42.5 c 122.3 g with >96% ee
99 28 97 97
96 24 97 77
a From chloroform-methanol (10:1). b By sublimation. c Not isolated.
(+)-1 with >97% ee, 39.8% yield, [R]20D +170 ( 20 (c ) 3.9, MeOH), mp 148-150 °C (racemic mp 203-206 °C). Solid (-)-1 was recrystallized in the same manner: total 122.3 g of (-)-1 with >96% ee, 35.7% yield, [R]20D -150 ( 20 (c ) 4.1, MeOH), mp 145-147 °C. The maximum yield in a resolution is 50%. Enantiomeric Purity. Enantiomers of 1 were separated on a Chiralcel OD HPLC column as reported previously.9 Enantiomers of 1, the four isomers of 1-monobutanoates and 1-dibutanoate were also separated on two Chiralpak AD HPLC columns connected in series (Chiral Technologies Inc. Exton, PA) eluted with hexanes-ethanol (92/8, 1 mL/min): (()-1dibutanoate: k′ ) 0.158; 1-5-monobutanoate: R ) 1.09, k′S ) 0.563, k′R ) 0.611; 1-4′-monobutanoate: R ) 1.11, k′R ) 0.755, k′S ) 0.838; 1: R ) 1.11, k′S ) 2.25, k′R ) 2.50. Enantiomers of the diesters did not separate on either the OD or the AD column. To determine the enantiomeric purity of 1-diesters, they were cleaved with methanol/sodium methoxide, and the enantiomeric purity of the resulting 1 was determined as above. Homology Model of the Open Confomation of CVL. We submitted the amino acid sequence of CVL (Swiss Prot code Q05489) and the open structure of PCL (Brookhaven protein databank code 1oil) to Swiss-Model,16 an automated protein modeling server, where the structure was modeled using ProMod 2.0. A calcium ion was added manually to the calcium ion site between Asp241 and Asp 287. The structure did not have any water molecules. A check of this model using the program WhatCheck17 revealed a number of side chain atoms in high-energy orientations, but the minimizations below corrected these errors. Modeling of Transition State Analogues in CVL. All modeling was done with Discover, version 2.9.7 (Biosym/MSI, San Diego, CA) using the Amber force field with a distance dependent dielectric constant of 4.0 and the 1-4 van der Waals interactions scaled by 50%. The distance dependent dielectric constant damps long-range electrostatic interactions to compensate for the lack of explicit solvation. Results were displayed using Insight II version 95.0 (Biosym/MSI). Protein structures in Figure 2 were created using RasMac v2.6.25 Using the Biopolymer module of Insight II, hydrogen atoms were added to correspond to pH 7.0. Histidines were uncharged, aspartates and glutamates were negatively charged and arginines and lysines were positively charged. The catalytic histidine (His286) was protonated. The phosphonate transition state analogue was built manually and covalently linked to Ser 87. Energy minimization proceeded in four stages. First, 200 of iterations steepest descent algorithm, all protein atoms constrained with a force constant of 10 kcal mol-1 Å-2; second, 200 iterations of conjugate gradients algorithm with the same constraints; and third, 500 iterations of conjugate gradients algorithm with only the backbone constrained by a 10 kcal mol-1 Å-2 force constant. For the fourth stage, minimization was continued using conjugate gradients algorithm without (25) Sayle, R. A.; Milner-White, E. J. Trends Biochem. Sci. 1995, 20, 374-376. http://www.umass.edu/microbio/rasmol/.
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any constraints until the rms deviation reached less than 0.005 Å mol-1. The five expected hydrogen bonds between the transition state analogue and the catalytic amino acid residues were similar for both the (R)- and (S)-enantiomers, but two of these hydrogen bonds were significantly longer than the expected 3.0 ( 0.2 Å. The N-O distances between the main chain amide of Gln88 and the oxyanion was 3.4-3.5 Å, and this distance between the imidazole and the O-aryl was 3.8 Å. We attribute these long bonds to either the lack of water
Gascoyne et al. molecules in the structure or inaccuracies in the homology model.
Acknowledgment. We thank the US Air Force for funding and NSERC (Canada) for the funds to purchase the modeling computer and software. We thank Michael (Xiaoming) Zhang for initial experiments with CRL. JO005681V
